ABCMdb

ABCC1 p.Arg1202Gly

Predicted by SNAP2:	A: D (85%), C: D (85%), D: D (95%), E: D (91%), F: D (91%), G: D (85%), H: D (66%), I: D (80%), K: D (85%), L: D (85%), M: D (85%), N: D (71%), P: D (95%), Q: D (85%), S: D (85%), T: D (80%), V: D (85%), W: D (85%), Y: D (85%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Localization of the GSH-dependent photolabelling s... Br J Pharmacol. 2003 Apr;138(8):1553-61. Ren XQ, Furukawa T, Aoki S, Sumizawa T, Haraguchi M, Che XF, Kobayashi M, Akiyama S
Localization of the GSH-dependent photolabelling site of an agosterol A analog on human MRP1.
Br J Pharmacol. 2003 Apr;138(8):1553-61., [PMID:12721111]

Abstract [show]
1. Human multidrug resistance protein 1 (MRP1) is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. We recently demonstrated that glutathione (GSH) is required for the labelling of the C-terminal half of MRP1 with a photoanalog of agosterol A (azido AG-A). In this study, we further characterized the GSH-dependent photolabelling site of azido AG-A on MRP1. 2. An epitope-inserted MRP1, MRP1 1222HA, which has two hemagglutinin A (HA) epitopes in the extracellular loop between transmembrane segment (TM) 16 and TM17 of the transporter, could bind azido AG-A in a GSH-dependent manner. 3. Protease digestion of the photolabelled MRP1 1222HA, followed by immunoprecipitation with an anti-HA antibody suggested that the GSH-dependent azido AG-A photolabelling site on MRP1 resides in the region within TM14-17 and the cytoplasmic region proximate to the C-terminus of TM17. 4. Arg(1210) in human MRP2 that corresponds to Arg(1202) in human MRP1 has an important role in the transporting activity of MRP2. Therefore, we replaced the Arg residue at position 1202 of MRP1 with Gly. Whereas photolabelling of the mutant MRP1 R1202G was greatly reduced, it retained leukotriene C(4) (LTC(4)) transport activity and conferred Vincristine resistance in LLC-PK1 cells. 5. In summary, this study demonstrated that the GSH-dependent azido AG-A photolabelling site on MRP1 resides in the region within TM14-17 and the cytoplasmic region proximate to the C-terminus of TM17. The charged amino acid Arg(1202) proximate to TM helix 16 is of critical importance for the GSH-dependent photolabelling of MRP1 with azido AG-A. Arg(1202) itself or the region nearby Arg(1202) may be involved in azido AG-A photolabelling.

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No. Sentence Comment
7 Whereas photolabelling of the mutant MRP1 R1202G was greatly reduced, it retained leukotriene C4 (LTC4) transport activity and conferred Vincristine resistance in LLC-PK1 cells. Login to comment
53 MRP1 constructs encoding R1202G was generated in PCR reaction using the forward primers: 50 -GGGCTCGAGTGT GTGGGCAACTGC-30 (bold denotes a mismatched base encoding the mutation). Login to comment
116 To investigate the role of the charged amino acid in the azido AG-A photolabelling of MRP1, we replaced the Arg1202 with Gly (R1202G). Login to comment
118 Figure 4a shows that the expression level of R1202G mutant was comparable to that of wild-type MRP1. Login to comment
119 The membrane vesicles expressing the wild-type and R1202G MRP1s were analyzed for their abilities to interact with azido AG-A in a GSH-dependent manner. Login to comment
120 As shown in Figure 4b, wild-type MRP1 was photolabelled with azido AG-A in a GSH-dependent manner; however, the GSH-dependent photolabelling of MRP1 R1202G with [125 I]azido AG-A was greatly reduced (Figure 4b). Login to comment
121 To test whether the mutation affects the function of MRP1, the LTC4 transport activity of MRP1 R1202G was examined. Login to comment
122 As shown in Figure 5a, MRP1 R1202G efficiently transported LTC4. Login to comment
123 The apparent Km values of MRP1 R1202G and wild-type MRP1 for LTC4 were 147 and 117 nM, respectively (Figure 5b). Login to comment
124 In order to test the ability of R1202G to confer drug resistance, the wild-type MRP1 and the MRP1 R1202G mutant cDNAs were cloned into the mammalian expression vector pCIneo and transfected into LLC-PK1 cells. Login to comment
139 Transfection of wild-type MRP1, or the MRP1 R1202G mutant, but not the pCIneo empty vector, resulted in drug-resistant colonies within 2 weeks. Login to comment
140 The drug resistance of stably transfected clones that express MRP1 or MRP1 R1202G (Figure 6a) was further tested in a cytotoxicity assay. Login to comment
141 As shown in Figure 6b, MRP1 and MRP1 R1202G conferred VCR resistance on LLC-PK1 cells. Login to comment
142 MRP1 R1202G mutant protein was localized to the plasma membrane in a similar manner to the wild-type MRP1, indicating that the R1202G mutation did not affect the trafficking of the mutant protein (Figure 6c). Login to comment
143 The fact that the photolabelling of MRP1 with azido AG-A was impaired by the R1202G mutation, but the transport activity and the ability to confer resistance to VCR were retained in this mutant suggests that the R1202G mutation affected only the photolabelling, but not the binding of azido AG-A to MRP1. Login to comment
144 To investigate whether the MRP1 R1202G mutant can interact with AG-A, the effect of AG-A on [3 H]LTC4 uptake by wild-type MRP1 and the MRP1 R1202G mutant expressed in Sf21 insect cells was examined. Login to comment
145 In the presence of 2 mM GSH, AG-A inhibited uptake of [3 H]LTC4 in membrane vesicles that express MRP1 R1202G in a dose-dependent manner and the extent of the inhibition was similar to that in membrane vesicles that express wild-type MRP1 (Figure 7). Login to comment
146 These findings suggest that MRP1 R1202G as well as wild-type MRP1 can interact with AG-A, and Arg1202 is not critical to the binding of AG-A to MRP1, although it affects the photolabelling of azido AG-A. Login to comment
177 When the membrane vesicles expressing the mutant protein were photolabelled and subsequently digested with trypsin or V8 Figure 4 Effect of R1202G mutation on the GSH-dependent photolabelling of MRP1 with [125 I]azido AG-A. Login to comment
178 (a) Wild-type MRP1 and MRP1 R1202G were expressed in insect cells. Login to comment
180 (b) Wild-type MRP1 and MRP1 R1202G membrane vesicles (100 mg of membrane proteins) were photolabelled with [125 I]azido AG-A in the absence or presence of the indicated concentrations of GSH as described in the legend to Figure 2a. Login to comment
182 Figure 5 Effect of R1202G mutation on ATP-dependent [3 H]LTC4 transport. Login to comment
183 (a) Time course of ATP-dependent transport of [3 H]LTC4 by wild-type MRP1 (square) and MRP1 R1202G (triangle) mutant. Login to comment
184 Membrane vesicles (50 mg) expressing wild-type MRP1 or MRP1 R1202G were incubated with 1.37 nM [3 H]LTC4 at 371C in 50 ml transport buffer as described in the legend to Figure 1b in the presence or absence of 4 mM ATP at the indicated periods. Login to comment
187 (b) Determination of the Km values of MRP1 wild type (square) and MRP1 R1202G (triangle) for [3 H]LTC4. Login to comment
205 On the other hand, if one of the trypsin digestion sites is located in the cytoplasmic portion Figure 6 Effect of R1202G mutation on drug resistance in LLC-PK1 cells. Login to comment
206 (a) Expression of wild-type MRP1 and the MRP1 R1202G mutant in LLC-PK1 cells. Login to comment
207 Crude membranes (50 mg of protein) prepared from LLC-PK1 cells transfected with either expression vectors encoding wild-type MRP1, MRP1 R1202G, or a control empty vector (pCIneo) were analyzed on 7.5% SDS - PAGE. Login to comment
210 LLC-PK1 cells expressing either wild-type MRP1 (square), MRP1 R1202G (triangle), or transfected with an empty vector (rhombus) were exposed to the indicated concentrations of VCR and the survival rate was determined by MTT assay as described under Materials and methods. Login to comment
213 Indirect immunofluorescent staining of wild-type MRP1 and MRP1 R1202G expressed in LLC-PK1 cells was carried out with the MRPm6 mAb and a FITC-conjugated secondary antibody. Login to comment
215 Both wild-type MRP1 and MRP1 R1202G were localized in the plasma membrane of LLC-PK1 cells. Login to comment
217 Figure 7 Effect of AG-A on [3 H]LTC4 transport by wild-type MRP1 and MRP1 R1202G. Login to comment
218 ATP-dependent [3 H]LTC4 uptake into MRP1 and MRP1 R1202G membrane vesicles from Sf21 insect cells was measured in the presence of 2 mM GSH and 10, 30 or 100 mM of AG-A as indicated. Login to comment
233 Therefore, we replaced the arginine at 1202 that is proximate to the N-terminus of TM16 of MRP1 with Gly (R1202G) to investigate the role of the charged amino acid in the region where GSH-dependent azido AG-A photolabelling site resides. Login to comment
234 GSH-dependent photolabelling of the mutant MRP1 R1202G with [125 I]azido AG-A was considerably lower than that of wild-type MRP1. Login to comment
235 The impaired GSH-dependent photolabelling of MRP1 R1202G may be attributable to the impaired binding of MRP1 to AG-A, and thus the GSH-dependent photolabelling with azido AG-A was lowered. Login to comment
241 Alternatively, the azido AG-A photolabelling site may reside in the region nearby Arg1202 that is proximate to the N-terminus of TM16, and the replacement of Arg1202 with Gly may affect the photolabelling of this site. Login to comment

[hide] Drug binding domains of MRP1 (ABCC1) as revealed b... Curr Med Chem Anticancer Agents. 2004 Jan;4(1):19-30. Karwatsky JM, Georges E
Drug binding domains of MRP1 (ABCC1) as revealed by photoaffinity labeling.
Curr Med Chem Anticancer Agents. 2004 Jan;4(1):19-30., [PMID:14754409]

Abstract [show]
Drug resistance is a major impediment in the treatment of cancer patients receiving single or multiple drug treatment. Efforts to reverse drug resistance of tumor cells have not been successful. In recent years, considerable emphasis has been placed on understanding the underlying mechanisms that confer drug resistance. The expression of the multidrug resistance protein 1 (MRP1 or ABCC1) in cancer cells has been shown to confer resistance to diverse classes of anti-cancer drugs. MRP1 is a member of the ATP-binding cassette (ABC) family whose function, in tumor cells, is to reduce drug accumulation through energized drug efflux. To learn more about the functions of MRP1 in tumor drug resistance, knowledge of the protein binding characteristics and the location of its binding sites are essential. Photoaffinity labeling (PAL) has emerged as a leading technique that can rapidly shed light on a protein's drug binding characteristics and ultimately drug binding domains. Several MRP1-specific photoreactive probes have been developed. PAL of MRP1 was first demonstrated with the quinoline-based drug, IAAQ. Other studies showed that the high affinity endogenous substrate of MRP1, LTC(4), has intrinsic photoreactive properties and binds within both N- and C-terminal domains of MRP1. LTC(4) is conjugated to glutathione (GSH), a property common to several MRP1 substrates. In addition, several unconjugated drugs have been identified that interact with MRP1: [(3)H]VF-13,159, IAAQ, IACI and IAARh123. Mapping studies showed that IACI and IAARh123 bind two sites within transmembrane (TM) regions 10-11 and 16-17 of MRP1. Interestingly, the GSH-dependent PAL of [(125)I]azidoAG-A and [(125)I]LY475776 occurs within, or proximal to TM 16-17. The PAL with several analogs of GSH, IAAGSH and azidophenacyl-[(35)S]GSH found to interact specifically with MRP1 within TM 10-11 and TM 16-17 in addition to binding two cytoplasmic regions in MRP1, L0 and L1. This review focuses on the use of PAL for studying MRP1 interactions with various drugs and cell metabolites. Furthermore, knowledge of MRP1 drug binding domains, as identified by PAL with various photoreactive drug analogs, provides an important first step towards more detailed analyses of MRP1 binding domains.

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No. Sentence Comment
199 PAL of MRP1 R1202G was greatly reduced, although it retained transport activity and conferred vincristine resistance. Login to comment

[hide] The MRP-related and BCRP/ABCG2 multidrug resistanc... Curr Drug Metab. 2004 Feb;5(1):21-53. Haimeur A, Conseil G, Deeley RG, Cole SP
The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation.
Curr Drug Metab. 2004 Feb;5(1):21-53., [PMID:14965249]

Abstract [show]
Several members of different families of the ATP-binding cassette (ABC) superfamily of transport proteins are capable of transporting an extraordinarily structurally diverse array of endo- and xenobiotics and their metabolites across cell membranes. Together, these transporters play an important role in the absorption, disposition and elimination of these chemicals in the body. In tumor cells, increased expression of these drug transporters is associated with resistance to multiple chemotherapeutic agents. In this review, current knowledge of the biochemical, physiological and pharmacological properties of nine members of the multidrug resistance protein (MRP)-related ABCC family (MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, MRP7, ABCC11 and ABCC12) as well as the G family member, ABCG2/BCRP, are summarized. A focus is placed on the structural similarities and differences of these drug transporters as well as the molecular determinants of their substrate specificities and transport activities. Factors that regulate expression of the MRP-related proteins and ABCG2/BCRP are also reviewed.

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No. Sentence Comment
276 Additional mutations of charged residues that lead to defective MRP1 transport activity (or altered drug interactions) have been described (e.g. TM16 Arg1202Gly and TM17 Arg1249Ala) but because only one or two substrates were tested in these studies, it is not possible to know whether the altered activity is relatively substrate selective or more global in nature [167, 168]. Login to comment

[hide] Mutational analysis of ionizable residues proximal... J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18. Situ D, Haimeur A, Conseil G, Sparks KE, Zhang D, Deeley RG, Cole SP
Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter.
J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18., 2004-09-10 [PMID:15208328]

Abstract [show]
The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4). In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding.

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No. Sentence Comment
4 In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. Login to comment
116 MRP1 expression levels in membrane vesicle proteins R1202G and R1202L prepared from cells expressing the neutrally substituted Arg1202 mutants were determined as described in A. Login to comment
118 Immunoblots of membrane vesicle proteins prepared from cells expressing the Glu1204 mutants E1204L and E1204D were carried out as described in A. TABLE I Summary of organic anion transport activity of MRP1 mutants with substitutions of ionizable amino acids in and proximal to TM13 to TM17 of MSD3 Mutation % Wild-type MRP1 transport activitya E217betaG LTC4 E1SO4 MTX GSH TM13 R1046D 115 70 80 120 NDb TM14 D1084R Ͻ10 Ͻ10 15 25 Ͻ10 D1084E 80 20 65 90 20 TM15 R1131E 70 50 80 60 ND TM16 R1197E Ͻ10 Ͻ10 Ͻ15 Ͻ10 ND R1197K 20 Ͻ25 Ͻ20 Ͻ10 ND R1202G 115 115 75 70 ND R1202L 115 120 50 110 ND E1204L Ͻ10 50 10 110 Ͻ25 E1204D 100 115 100 115 Ͻ25 TM17 R1249D Ͻ10 Ͻ15 Ͻ10 Ͻ10 ND R1249K Ͻ10 10 Ͻ15 Ͻ10 ND a The values shown are means of duplicate or triplicate determinations and are derived from Fig. 2, 4, and 5 (see figure legends for details). Login to comment
124 Immunoblots showed that expression levels of the R1202G and R1202L mutants (Fig. 3B) and the E1204L and E1204D mutants (Fig. 3C) ranged from 80 to 225% of wild-type MRP1. Login to comment
127 At 1 min, E217betaG and LTC4 uptake by the R1202G and R1202L mutants was comparable with wild-type levels (Fig. 4, A and B). Login to comment
128 Levels of E1SO4 uptake by the R1202G mutant were reduced by ϳ25%, whereas uptake by the R1202L mutant was reduced by ϳ50% (Fig. 4C). Login to comment
129 Uptake levels of MTX by the R1202G mutant was moderately reduced (by ϳ30%), whereas uptake by R1202L was comparable with wild-type MRP1 (Fig. 4D). Login to comment
169 A-D, uptake of 3 H-labeled organic anions by the membrane vesicles shown in Fig. 3B which were prepared from cells transfected with empty pcDNA3.1 vector (open bars), and vector containing wild-type MRP1 cDNA (black bars), and the neutrally substituted Arg1202 mutant R1202G and R1202L cDNAs (gray bars). Login to comment
201 In addition to being readily expressed, the neutrally substituted mutants of TM16 Arg1202 (R1202G and R1202L) exhibited transport activities that were, in the case of most substrates, similar to those of wild-type MRP1. Login to comment
202 Our findings are consistent with a recent report that a neutrally substituted Arg1202 mutant (R1202G) could still transport LTC4 (28). Login to comment